Proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are classed as belonging to the serine proteases, owing to the catalytically active amino acids. They are naturally produced and secreted by microorganisms, in particular by Bacillus species. They act as unspecific endopeptidases, i.e. they hydrolyze any acid amide bonds located inside peptides or proteins. Their pH optimum is usually within the distinctly alkaline range. A review of this family is provided, for example, by the paper “Subtilases: Subtilisin-like Proteases” by R. Siezen, pages 75-95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, New York, 1996. Subtilisins are suitable for a multiplicity of possible technical uses, as components of cosmetics and, in particular, as active ingredients of detergents or cleaning agents.
Apart from other enzymes such as, for example, amylases, lipases or cellulases, proteases are used as active components in detergents and cleaning agents. They have the ability to break down proteinaceous soilings on the material to be cleaned such as, for example, textiles or dishes. Owing to their relatively high solubility, the hydrolysis products are washed away with the wash liquor or are attacked, dissolved, emulsified or suspended by the other components of the detergents or cleaning agents. Thus, synergistic effects between the enzymes and the other components of the detergents and cleaning agents in question can arise. Owing to their favorable enzymic properties such as stability or pH optimum, subtilisins stand out among the detergent and cleaning agent proteases. The most important ones and the most important strategies for their technical development are stated below.
The fundamental strategy for developing detergent proteases is to first isolate microbially and naturally produced enzymes and to test them for their principle suitability for this possible use. These molecules may then be optimized. Thus, for example, the protease 164-A1 from Chemgen Corp., Gaithersburg, Md., USA, and Vista Chemical Company, Austin, Tex., USA, obtainable from Bacillus spec. 164-A1, is suitable for use in detergents and cleaning agents, according to the application WO 93/07276 A1. Other examples are alkaline protease from Bacillus sp. PD138, NCIMB 40338 from Novozymes (WO 93/18140 A1), the proteinase K-16 from Kao Corp., Tokyo, Japan, derived from Bacillus sp. ferm. BP-3376 (U.S. Pat. No. 5,344,770) and, according to WO 96/25489 A1 (Procter & Gamble, Cincinnati, Ohio, USA), the protease of the psychrophilic organism Flavobacterium balustinum. 
Subtilisin BPN′ which is derived from Bacillus amyloliquefaciens, and B. subtilis, respectively, has been disclosed in the studies by Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 and by J. A. Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925. Subtilisin BPN′ serves as reference enzyme of the subtilisins, in particular with respect to numbering of positions. Thus, for example, the point mutations of the application EP 130756 A1 which refer to all subtilisins are also indicated with BPN′ numbering. These merely include position 217 which corresponds to position 211 in enzymes of the invention; no particular substitution is specifically emphasized for this; all of them are mentioned, except replacement with M, W, C or K; preference should be given to that with A or S.
The application CA 2049097 A1 studies multiple mutants of this molecule, in particular with respect to their stability in detergents and cleaning agents. These include variants containing the substitutions Y217K and Y217L and also the double mutant S63D/Y217K, i.e. those containing substitutions which correspond to positions 211 and, respectively, 61 and 211 of B. lentus alkaline protease. However, no amino acids corresponding to any of the proteases of the present application at these positions were introduced.
Variants obtained by point mutations in the loop regions of said enzyme and having reduced binding to the substrate with a simultaneously increased rate of hydrolysis are introduced, for example, in the patent applications WO 95/07991 A2 and WO 95/30010 A1. WO 95/07991 A2 relates to the sixth loop of the molecule and discloses double mutants in which, in addition to another mutation, the amino acids at position 217 (corresponding to 211 in B. lentus alkaline protease) has been mutated to D, for example. Since BPN′ by nature has I at position 205 (corresponding to 199), these two positions at most may be regarded herein as having been described previously, but always in combination with other mutations in subtilisin loop regions and with specific changes in the enzymic properties. The patent application WO 95/29979 A1, for example, discloses detergents containing BPN′ variants of this kind. WO 95/30010 A1 discloses further mutations in the other five loop regions, including at position 63 (corresponding to 61), but only to D or E at this position. In contrast, two of the amino acid positions considered in the present patent application, namely positions 3 and 4, are not located in loop regions. On the other hand, the numerous substitutions indicated in said documents do not correlate with stabilizations, in particular with stabilizing mutations of subtilisin BPN′.
The publications by E. L. Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191, and by Jacobs et al. (1985) in Nucl. Acids Res., Volume 13, pp. 8913-8926 introduce the protease subtilisin Carlsberg. It is naturally produced by Bacillus licheniformis and was and, respectively, is obtainable under the trade name Maxatase® from Genencor International Inc., Rochester, N.Y., USA, and under the trade name Alcalase® from Novozymes A/S, Bagsvaerd, Denmark. Variants thereof which are obtainable by point mutations and have reduced binding to the substrate with a simultaneously increased rate of hydrolysis are disclosed, for example, by the application WO 96/28566 A2. These are variants in which single or multiple substitutions in the loop regions of the molecule have been carried out. The only variants having substitutions at positions corresponding to those of the present application, which have been tested in washing or cleaning experiments, are those of multiple mutants which have among other substitutions those of G62 (corresponding to position 61 of B. lentus alkaline protease) by N, D, Q, E, P or S, but not by A, of V204 (corresponding to position 199) by various other amino acids, but not by I, and of L216 (corresponding to position 211) by 14 other amino acids, including also by D. Thus, the only variations relating to the present application, which have been described previously by this document, are 3T—because T naturally occupies position 3 in subtilisin Carlsberg—and 211D.
The protease PB92 is produced naturally by the alkaliphilic bacterium Bacillus nov. spec. 92 and was obtainable under the trade name Maxacal® from Gist-Brocades, Delft, The Netherlands. Its original sequence is described in patent application EP 283075 A2. Variants of said enzyme which have been obtained by point mutation and which are suitable for use in detergents and cleaning agents are disclosed in the applications WO 94/02618 A1 and EP 328229 A1, for example. The first of said applications describes only substitutions at position 211, by various amino acids, but not by D. The second document discloses that particular regions in whch the two residues 61 and 211 are also present are involved in substrate binding. However, 61 is not listed among the positions particularly interesting for mutagenesis, and a substitution by Y is proposed for 211, which is able to increase the washing performance of a corresponding formulation only in combination with at least one further substitution, however.
The subtilisins 147 and 309 are sold by Novozymes under the trade names Esperase® and Savinase®, respectively. They are originally derived from Bacillus strains disclosed by the application GB 1243784 A. Variants of said enzymes, which have been developed by means of point mutagenesis with respect to usage in detergent and cleaning agents, are disclosed, for example, in the applications WO 94/02618 A1 (see above), WO 89/06279 A1, WO 95/30011 A2 and WO 99/27082 A1.
The application WO 89/06279 A1 aimed at achieving higher oxidation stability, an increased rate of proteolysis and enhanced washing performance. It reveals that substitutions at particular positions should alter the physical or chemical properties of subtilisin 147 or 309 molecules (whose numbering corresponds to that of Bacillus lentus DSM 5483 alkaline protease); among said positions, mention is made of, inter alia, position 199, but no special substitution is described. The application WO 95/30011 A2 introduces variants of subtilisin 309 which have point mutations in the loop regions of the molecule and thus exhibit reduced adsorption to the substrate with a simultaneously increased rate of hydrolysis. The positions 61, 199 and 211 are also present in such regions. The substitution L211D, inter alia, is proposed for position 211 therein; the substitutions of G by N, D, Q, E, P or S are proposed for positions 61, with numerous substitutions, but not I, being proposed for 199. The application WO 99/27082 A1 develops variants of, by way of example, subtilisin 309, whose washing performance is enhanced by enlarging the active loops by inserting at least one amino acid. Thus, they are not substitutions like in the present application.
Subtilisin DY has originally been described by Nedkov et al. 1985 in Biol. Chem Hoppe-Seyler, Volume 366, pp. 421-430. According to the application WO 96/28557 A2, for example, it may be optimized via specific point mutations in the active loops for usage in detergents and cleaning agents, producing variants having reduced adsorption and an increased rate of hydrolysis, including those containing substitutions at position 62 (corresponding to 61 in B. lentus alkaline protease) of G by N, D, Q, E, P or S, at position 204 (corresponding to 199), but not 2041, and at position 216 (corresponding to 211) numerous substitutions, including also D. Since subtilisin DY by nature has T at position 3, only a variant 3T/211D has at most been previously described hereby.
The enzyme thermitase produced naturally by Thermoactinomyces vulgaris has originally been described by Meloun et al. (FEBS Lett. 1983, pp. 195-200). The application WO 96/28558 A2, for example, discloses variants having reduced absorption and an increased rate of hydrolysis, owing to substitutions in the loop regions. There, substitutions at position 211 (corresponding to 211 in B. lentus alkaline protease) by 14 amino acids, including also D, and at position 70 (corresponding to 61), of G by N, D, Q, E, P or S are described. Since I is naturally present at position 209 of thermitase (corresponding to 199), this suggests at most the variants 1991 and 211D of the proteases essential to the present application. In particular it also does not suggest any stabilizations, for example by threonine at position 3 and/or isoleucine at position 4 (according to B. lentus alkaline protease). At the correspondingly, homologous positions 10 and 11, thermitase has the amino acids S and R (compare alignment in WO 91/00345 A1). Moreover, thermitase is a molecule whose sequence overall deviates considerably from those of the other subtilisins. Thus the homology between the mature proteins thermitase and B. lentus DSM 5483 alkaline protease (see below) is 45% identity (62% similar amino acids).
Proteinase K is also a protease which has comparatively low homology to B. lentus alkaline protease. Said homology is only 33% identity (46% similar amino acids) at the mature protein level. Proteinase K is originally from the microorganism Tritirachium album Limber and has been described by K.-D. Jany and B. Mayer 1985 in Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 485-492. WO 88/07581 A1 discloses the very similar proteases TW3 and TW7, inter alia for usage in detergents and cleaning agents. The application WO 96/28556 A2 describes numerous substitutions in proteinase K, including at position 220 (corresponding to 211 in B. lentus alkaline protease) by 14 other amino acids, including also D, and at position 68 (corresponding to 61) of G by N, D, Q, E, P or S. Since proteinase K has by nature I at position 208 (corresponding to 199) and T at position 4 (corresponding to 3), this suggests at most the variations 3T, 199I and 211D of the proteases essential to the present application.
Finally, mention should also be made of Bacillus subtilis bacillopeptidase F which by nature has the amino acids alanine and isoleucine at positions 61 and 199, respectively. Otherwise, however, it has only low similarity to protease variants of the invention: at the amino acid level, only a homology of 30% identity, or 38% of similar amino acids, can be found. This enzyme is listed in the abovementioned work by Siezen et al., but up until now has not been described or claimed yet for usage in detergents and cleaning agents.
The applications EP 199404 A2, EP 251446 A1, WO 91/06637 A1 and WO 95/10591 A1, for example, describe further proteases which are referred to by Procter & Gamble Comp., Cincinnati, Ohio, USA as “protease A”, “protease B”, “protease C” and “protease D”, respectively, and which are suitable for technical use, in particular in detergents and cleaning agents. The proteases of the application EP 199404 are various BPN′ variants which are based on the application EP 130756 A1 (see above), but which have no variations at the positions relevant to the present application. EP 251446 A1 discloses numerous BPN′ variants, including also 217-variants (corresponding to position 211 in B. lentus alkaline protease); any possible substitutions are mentioned here, not disclosing, however, which properties accompany the variation 217D. According to the application WO 91/06637 A1, “proteases C” are distinguished by point mutations of BPN′ at positions 123 and/or 274. “Protease D” comprises variants, primarily of Bacillus lentus protease, which, according to WO 95/10591 A1, carry mutations at position 76 (according to BPN′ numbering, corresponding to position 74 in B. lentus alkaline protease) and, in addition at other positions. The latter may also include position 217 (corresponding to 211); however, no substitution by D has been previously described therein. Virtually the same also applies to U.S. Pat. No. 6,017,871 A, for example, for usage in detergents and cleaning agents and cosmetics and to U.S. Pat. No. 5,677,272 A and U.S. Pat. No. 6,066,611 A, for example, for usage in bleaches: there, the substitution 217D is also mentioned in principle, again in combination with the substitution at position 76, but is not preferred.
Other known proteases are the enzymes obtainable under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase® and Kannase® from Novozymes, under the trade names Maxapem®, Purafect®, Purafect OxP® and Properase® from Genencor, under the trade name Protosol® from Advanced Biochemicals Ltd., Thane, India and under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China.
One strategy for enhancing the washing performance of subtilisins is to introduce randomly or specifically point mutations into the known molecules, owing to known functions of individual amino acids, and to test the variants obtained for their washing performance contributions. This strategy is pursued, for example, by U.S. Pat. No. 5,700,676 and the application EP 130756 A1 (see above). The only position described therein which relates to the present invention is a substitution at position 217 (corresponding to 211 in B. lentus alkaline protease) by any of the 19 amino acids, either alone or in addition to other substitutions which, however, are not relevant to the present application. The same also applies to U.S. Pat. No. 5,801,038. U.S. Pat. No. 5,441,882 describes the method of modifying the enzymic properties via particular single substitutions, including also at position 217 (corresponding to 211 in B. lentus alkaline protease), either alone or in addition to other substitutions which, however, are not relevant to the present application. U.S. Pat. No. 4,760,025 discloses corresponding variants which, however, contain in each case only one substitution; included here is again only position 217 and without disclosure of a concrete substitution therefor.
In order to enhance the washing performance of subtilisins, numerous applications pursued the strategy of inserting additional amino acids into the active loops, thus, for example, apart from the already mentioned WO 99/27082 A1, also the applications published with the numbers WO 00/37599 A1, WO 00/37621 A1 to WO 00/37627 A1 and WO 00/71683 A1 to WO 00/71691 A1. Said strategy should accordingly be applicable in principle to all subtilisins belonging to either of the subgroups I-S1 (true subtilisins) or I-S2 (highly alkaline subtilisins).
Another strategy of enhancing the performance is to modify the surface charges and/or the isoelectric point of the molecules, thereby altering their interaction with the substrate. Variations of this kind are introduced, for example, by U.S. Pat. No. 5,665,587 and the applications EP 405901 A1 and WO 91/00334 A1. Numerous positions are mentioned therein, including in each case also 3, 4 and 217 (corresponding to 3, 4 and 211 in B. lentus alkaline protease), but without actually disclosing corresponding variants. The application WO 91/00345 A1 also refers to these positions for the same purpose, likewise without actually indicating corresponding variants. WO 92/11348 A1 discloses point mutations for reducing the pH-dependent variation in the molecular charge. This may at most relate to the substitutions S3T and L211D which characterize the present application; however, no relevant substitution is directly disclosed therein. The application WO 00/24924 A2 derives from this principle a method for identifying variants which are supposedly suitable for usage in detergents and cleaning agents; all variants disclosed here have at least one substitution at position 103, preference being given to multiple variants containing no substitution relevant to the present application. According to WO 96/34935 A2, it is also possible to increase the hydrophobicity of the molecules for the purpose of enhancing the performance in detergents and cleaning agents, and this may influence the stability of the enzyme.
The application WO 99/20727 A2 discloses subtilisin variants as may have been obtained by a method of the application WO 00/24924 A2: they all comprise at least one substitution at position 103, combined with a multiplicity of other possible substitutions, none of them, however, at the position corresponding to position 61 of B. lentus protease. Preference is given to multiple variants having at least six substitutions, including also positions 205 and 217 (corresponding to 199 and 211 in B. lentus alkaline protease); only two of more than 50 of said variants actually have the substitution 199I relevant to the present application. The applications WO 99/20723 A2 and WO 99/20726 A2 disclose the same mutants for detergents and cleaning agents which additionally contain an amylase, or bleach.
A modern direction in enzyme development is to combine, via statistical methods, elements from known proteins related to one another to give novel enzymes having properties which have not been achieved previously. Methods of this kind are also listed under the generic term directed evolution and include, for example, the following methods: the StEP method (Zhao et al. (1998), Nat. Biotechnol., Volume 16, pp. 258-261), random priming recombination (Shao et al., (1998), Nucleic Acids Res., Volume 26, pp. 681-683), DNA shuffling (Semmer, W. P. C. (1994), Nature, Volume 370, pp. 389-391) or RACHITT (Coco, W. M. et al. (2001), Nat. Biotechnol., Volume 19, pp. 354-359).
Another, in particular complementary, strategy is to increase the stability of the proteases concerned and thus to increase their efficacy. For example, U.S. Pat. No. 5,230,891 has described stabilization via coupling to a polymer for proteases used in cosmetics; said stabilization is accompanied by enhanced skin compatibility. Especially for detergents and cleaning agents, on the other hand, stabilizations by point mutations are more familiar. Thus, according to U.S. Pat. No. 6,087,315 and U.S. Pat. No. 6,110,884, it is possible to stabilize proteases by replacing particular tyrosine residues with other residues. WO 89/09819 A1 and WO 89/09830 A1 describe relatively thermostable BPN′ variants which have at positions 217 (corresponding to 211 in B. lentus alkaline protease) substitutions by K or L and, in addition to 217K, the substitution S63D at position 63 (corresponding to position 61).
Other possible examples of stabilization via point mutagenesis, which have been described, are 1) replacing particular amino acid residues with proline according to WO 92/19729 A1, and, respectively, EP 583339 B1 and U.S. Pat. No. 5,858,757 and according to EP 516200 A1; 2) introducing more polar or more highly charged groups on the molecule surface, according to EP 525610 A1, EP 995801 A1 and U.S. Pat. No. 5,453,372, inter alia at the position corresponding to V4 of B. lentus protease; in contrast, the exchange V4I, as in the present application, introduces a less polar amino acid; 3) enhancing the binding of metal ions, in particular via mutagenesis of calcium binding sites, for example according to the teaching of the applications WO 88/08028 A1 and WO 88/08033 A1; or 4) blocking autolysis by modification or mutagenesis, for example according to U.S. Pat. No. 5,543,302.
The application EP 398539 A1 discloses a combination of two or more stabilization strategies. Accordingly, subtilisins may be stabilized and their contribution to the washing or cleaning performance may be improved by (1.) replacing amino acids of the calcium binding sites with more negative ones, (2.) deleting or mutating natural Asn-Gly sequences, (3.) replacing Met residues with other residues and (4.) additionally substituting particular amino acids close to the catalytic center. None of the first three possibilities applies to the variants of the invention of the present application. The fourth possibility relates to positions 61 and 211. Here it is suggested, however, to replace the amino acids naturally present at these positions (S63 and Y217 in subtilisin BPN′) with G and L, respectively. In contrast, these positions in particular are occupied by amino acids other than G or L in the molecules of the present application.
Further possibilities of stabilizing subtilisins, in particular those derived from that of Bacillus lentus, via point mutagenesis are reported in U.S. Pat. No. 5,340,735, U.S. Pat. No. 5,500,364, U.S. Pat. No. 5,985,639 and U.S. Pat. No. 6,136,553. The mutated positions are determined via analysis of the three dimensional structure. Variants at positions 61 and 211, however, are described in none of these documents.
The documents EP 755999 A1 and WO 98/30669 A1, for example, disclose that proteases, in particular performance-enhanced proteases, may be used together with α-amylases and other detergent enzymes in detergents and cleaning agents in order to enhance the washing or cleaning performance. The application WO 97/07770 A1, for example, discloses that some of those which have previously been established as detergent proteases (see below) are also suitable for cosmetic purposes. The application EP 380362 A1, for example, introduces another possible use of proteases, which relates to organochemical syntheses for which, according to said application, those subtilisins should be suitable which have been stabilized via point mutagenesis at, according to B. lentus alkaline protease numbering, positions 61 (by mutation to D) and/or 211 (by mutation to K or L), either alone or in addition to other mutations. Thus, in this connection too, no substitution relevant to the present invention has been described previously.
The B. lentus alkaline proteases are highly alkaline proteases of Bacillus species. According to the application WO 91/02792 A1, one of these strains has been deposited under number DSM 5483; the sequences and biochemical properties of the wild-type enzyme are also disclosed therein. WO 92/21760 A1 and WO 95/23221 A1 disclose variants of this enzyme, to be obtained by point mutation and suitable for use in detergents and cleaning agents.
The wild-type enzyme is derived from a producer which had originally been obtained by screening for alkaliphilic Bacillus strains and displayed itself a comparatively high stability to oxidation and the action of detergents. The applications WO 91/02792 A1 and, respectively EP 493398 B1 and U.S. Pat. No. 5,352,604 describe its heterologous expression in the host Bacillus licheniformis ATCC 53926. The claims of said US patent refer to positions 208, 210, 212, 213 and 268 as being characteristic for B. lentus alkaline protease; said positions correspond to positions 97, 99, 101, 102 and 157 in the numbering of the mature protein, in which positions this enzyme differs from the mature Savinase®. The three dimensional structure of this enzyme is described in the publication Goddette et al. (1992), J. Mol. Biol. , Volume 228, pp. 580-595: “The crystal structure of the Bacillus lentus alkaline protease, Subtilisin BL, at 1.4 Å resolution”.
The application WO 92/21760 A1, or U.S. Pat No. 5340735, also discloses the amino acid sequence, under SEQ ID No. 52 therein, and the nucleotide sequence, under SEQ ID No. 106 therein, of the B. lentus alkaline protease wild-type enzyme produced by B. lentus DSM 5483. In addition, this application discloses 51 different variants derived from said protease, which differ from the wild type in single or in each two or more positions from the wild type and which thereby have been stabilized. Said variants also include the substitutions S3T, V4I and VI 199I. According to this application, most preference is given to the variant M131 containing the substitutions S3T/V4I/A188P/V193M/V199I which has been deposited under the reference ATCC 68614 with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA. This variant serves as starting enzyme for the present application (see Example 1) and its DNA sequence and amino acid sequence are also indicated in the sequence listing under SEQ ID Nos 1 and 2, respectively. All of these variants are thus derived from Bacillus lentus DSM 5483 alkaline protease. The US patents U.S. Pat. No. 5,500,364 and U.S. Pat. No. 5,985,639 derived from the WO document, for example, disclose variants whose stability has been enhanced by point mutations at different positions.
The application WO 95/23221 A1 reveals B. lentus alkaline protease variants whose performance for usage in detergents and cleaning agents has been enhanced by specific point mutagenesis and which are to be considered as further developments of the aforementioned molecules. Some of those likewise have the three substitutions S3T, V4I and V199I. In addition, they all have two or three further point mutations compared to the wild-type enzyme from B. lentus DSM 5483. Some of them carry an additional mutation at position 211, namely 211D (variant F49, F54 and F55). Consequently, said application, and the corresponding U.S. Pat. No. 5,691,295, U.S. Pat. No. 5,801,039 and U.S. Pat. No. 5,855,625 claim variants containing the substitutions 211D and 211E. U.S. Pat. No. 6,197,589 illustrates the corresponding strategy, namely to specifically modify the charge conditions close to the substrate binding pocket.
As all of these studies which have been carried out over a long period of time confirm, there is high demand for technically useable proteases some of which differ drastically, some only in a few positions, from previously known proteases. They cover thus a broad spectrum of very drastic, down to very subtle performance differences. This is evident especially in their use in detergents and cleaning agents. During their development, the behavior of said enzymes, for example in the context of a detergent or cleaning agent formulation, cannot be readily inferred from the possibly calculable enzymic properties. Other factors such as stability toward high temperatures, oxidizing agents, denaturation by surfactants, folding effects or desired synergies with other ingredients play a part here and can frequently be determined only experimentally.